A growing number of diseases, including cystic fibrosis, Alzheimer~s and scrapie, appear to be caused at the molecular level by errors in protein folding. These errors involve either, non-productive, folding or pathogenic molecular aggregation which results from partial unfolding. For this reason, a molecular understanding of the mechanisms of protein folding is more important than ever before. A simple calculation can show that the number of possible conformations available to a small protein is astronomically large and impossible to sample in the length of time it takes to fold. Thus, the sequence of a protein must not only dictate its final folded state, but also the path by which it folds, thereby eliminating the necessity to sample all possible folded conformations. The fastest folding proteins are ones which have well defined folding pathways that guide them efficiently, without detour to the native folded state. Dr. Oas has developed a version (16- 85) of the N-terminal domain of phage lambda repressor, which is monomeric, stable, and forms the same structure as a longer version bound to DNA. His previous studies have shown that the G46A/G48A variant of 16-18 folds in 20 microsec, almost 1000 times faster than commonly reported fast protein folding rates. The work described in this proposal is aimed at understanding the fundamental steps involved in the rate limiting step for 16-85. This will necessistate characterization of the conformational and energetic properties of the three experimentally accessible forms of the protein: the native (N), denatured (D) and transition (T) states. The experimental approach involves making gly-ala mutational variants of 16-85 and studying the effect of these mutations on N, D and T. The primary analytical tool will be nuclear magnetic resonance (NMR), which will be used to measure amide hydrogen exchange rates and backbone dynamics of N; translational diffusion rates and chemical shifts in both N and D; and finally the rates of folding and unfolding and their sensitivity to mutation and urea to characterize T. In addition to NMR, Dr. Oas will use circular dichroism-based denaturation surfaces to determine the thermodynamic properties of the N-D equilibrium. Most of these methods have been shown to work well with wild type 16-85 and several variants. He therefore anticipates that he will be able to thoroughly characterize the 16-85 folding pathway in the proposed three year grant period.